This project will elucidate key aspects of the interactions between the thin filament and myosin in muscle. Innovative experimental approaches are developed to address specific current hypotheses in this research. The questions examined include 1) does the lever arm of myosin swing from the M line toward the Z line of muscle? 2) what structural changes occur to the thin filament during myosin binding? and 3) how is the flexibility of myosin heads on the thick filament affected by nucleotide binding and light chain phosphorylation? Experimental designs include the development of an actomyosin nanocircuit to detect structural changes in the myosin head only when it is in brief contact with actin. Novel relays of time-resolved fluorescence resonance energy transfer probes are employed to determine precise event-correlated distance measurements. Spectroscopically-assisted docking simulations and computational analysis of the atomic models of myosin and the thin filament will provide a more detailed view of their interface that will include known regulatory proteins on the thin filament and their intricate interplay with actomyosin interactions. To more carefully examine the constraints on myosin heads orientations when attached to actin, a new single molecule assay is proposed that enables the direct observation of myosin head twisting relative to the thick filament backbone. Total internal reflectance fluorescence microscopy combined with imaging through intensified CCD and digital image analysis will reveal functional effects of structural perturbations to this critical region in the myosin molecule. The results of these studies will clarify important issues in muscle force generation and its regulation that require a thorough understanding to develop successful therapeutic and public health strategies to combat cardiovascular diseases. For instance, a number of genetic mutations in sarcomeric proteins, especially beta-cardiac myosin, are known to induce familial hypertrophic cardiomyopathies. These diseases are among the most common causes of cardiac arrest in young adults. The biological basis of these diseases and the identification of all genetic alterations that lead to the disease would be greatly clarified if the molecular mechanisms of muscle contraction were understood at the atomic level. Recent progress being made toward achieving such an understanding of this critical process in motility and the proposed research will contribute to this effort.